scholarly journals The roles of Ca2+ and plasma membrane ion channels in hyphal tip growth of Neurospora crassa

1995 ◽  
Vol 108 (11) ◽  
pp. 3405-3417 ◽  
Author(s):  
N.N. Levina ◽  
R.R. Lew ◽  
G.J. Hyde ◽  
I.B. Heath
Keyword(s):  
Plasma Membrane ◽  
Neurospora Crassa ◽  
Tip Growth ◽  
Apical Plasma Membrane ◽  
Pipette Solution ◽  
Saprolegnia Ferax ◽  
High Gradients ◽  
Voltage Clamping ◽  
Stretch Activated Channels ◽  
Hyphal Tip

Growing hyphae of the ascomycete fungus Neurospora crassa contained a tip-high gradient of cytoplasmic Ca2+, which was absent in non-growing hyphae and was insensitive to Gd3+ in the medium. Patch clamp recordings in the cell-attached mode, from the plasma membrane of these hyphae, showed two types of channel activities; spontaneous and stretch activated. The spontaneous channels were identified as inward K+ channels based on inhibition by tetraethylammonium. The stretch activated channels had increased amplitudes in response to elevated Ca2+ in the pipette solution, and thus are permeable to Ca2+ and mediate inward Ca2+ movement. Gd3+, which is an inhibitor of some stretch activated channels, incompletely inhibited stretch activated channel activity. Both tetraethylammonium and Gd3+ only transiently reduced the rates of tip growth without changing tip morphology, thus indicating that the channels are not absolutely essential for tip growth. Furthermore, in contrast to the hyphae of another tip growing organism, Saprolegnia ferax, tip-high gradients of neither spontaneous nor stretch activated channels were found. Voltage clamping of the apical plasma membrane potential in the range from -300 to +150 mV did not affect the rates of hyphal elongation. Collectively, these data suggest that ion transport across the plasma membrane at the growing tip in Neurospora is not obligatory for the maintenance of tip growth, but that a gradient of Ca2+, possibly generated from internal stores in an unknown way, is required.

Mechanisms of Hyphal Tip Growth and Morphogenesis in the Filamentous Fungus Neurospora crassa

2004 ◽  
Vol 10 (S02) ◽  
pp. 1554-1555
Author(s):  
Maho Uchida ◽  
Solomon Bartnicki-García ◽  
Robert W. Roberson
Keyword(s):  
Neurospora Crassa ◽  
Filamentous Fungus ◽  
Tip Growth ◽  
Hyphal Tip Growth ◽  
Hyphal Tip

Extended abstract of a paper presented at Microscopy and Microanalysis 2004 in Savannah, Georgia, USA, August 1–5, 2004.

scholarly journals The Neurospora crassa exocyst complex tethers Spitzenkörper vesicles to the apical plasma membrane during polarized growth

2014 ◽  
Vol 25 (8) ◽  
pp. 1312-1326 ◽  
Author(s):  
Meritxell Riquelme ◽  
Erin L. Bredeweg ◽  
Olga Callejas-Negrete ◽  
Robert W. Roberson ◽  
Sarah Ludwig ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Neurospora Crassa ◽  
Hyphal Growth ◽  
Apical Plasma Membrane ◽  
Apical Region ◽  
Secretory Vesicles ◽  
Polarized Growth ◽  
Hyphal Morphogenesis ◽  
Exocyst Complex ◽  
Dynamic Formation

Fungal hyphae are among the most highly polarized cells. Hyphal polarized growth is supported by tip-directed transport of secretory vesicles, which accumulate temporarily in a stratified manner in an apical vesicle cluster, the Spitzenkörper. The exocyst complex is required for tethering of secretory vesicles to the apical plasma membrane. We determined that the presence of an octameric exocyst complex is required for the formation of a functional Spitzenkörper and maintenance of regular hyphal growth in Neurospora crassa. Two distinct localization patterns of exocyst subunits at the hyphal tip suggest the dynamic formation of two assemblies. The EXO-70/EXO-84 subunits are found at the peripheral part of the Spitzenkörper, which partially coincides with the outer macrovesicular layer, whereas exocyst components SEC-5, -6, -8, and -15 form a delimited crescent at the apical plasma membrane. Localization of SEC-6 and EXO-70 to the plasma membrane and the Spitzenkörper, respectively, depends on actin and microtubule cytoskeletons. The apical region of exocyst-mediated vesicle fusion, elucidated by the plasma membrane–associated exocyst subunits, indicates the presence of an exocytotic gradient with a tip-high maximum that dissipates gradually toward the subapex, confirming the earlier predictions of the vesicle supply center model for hyphal morphogenesis.

scholarly journals Structure and Distribution of Organelles and Cellular Location of Calcium Transporters in Neurospora crassa

2009 ◽  
Vol 8 (12) ◽  
pp. 1845-1855 ◽  
Author(s):  
Barry J. Bowman ◽  
Marija Draskovic ◽  
Michael Freitag ◽  
Emma Jean Bowman
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Neurospora Crassa ◽  
Fluorescent Protein ◽  
Protein A ◽  
Vacuolar Membrane ◽  
Red Fluorescent Protein ◽  
Animal Cells ◽  
Marker Proteins ◽  
Hyphal Tip

ABSTRACT We wanted to examine the cellular locations of four Neurospora crassa proteins that transport calcium. However, the structure and distribution of organelles in live hyphae of N. crassa have not been comprehensively described. Therefore, we made recombinant genes that generate translational fusions of putative organellar marker proteins with green or red fluorescent protein. We observed putative endoplasmic reticulum proteins, encoded by grp-78 and dpm, in the nuclear envelope and associated membranes. Proteins of the vacuolar membrane, encoded by vam-3 and vma-1, were in an interconnected network of small tubules and vesicles near the hyphal tip, while in more distal regions they were in large and small spherical vacuoles. Mitochondria, visualized with tagged ARG-4, were abundant in all regions of the hyphae. Similarly, we tagged the four N. crassa proteins that transport calcium with green or red fluorescent protein to examine their cellular locations. NCA-1 protein, a homolog of the SERCA-type Ca2+-ATPase of animal cells, colocalized with the endoplasmic reticulum markers. The NCA-2 and NCA-3 proteins are homologs of Ca2+-ATPases in the vacuolar membrane in yeast or in the plasma membrane in animal cells. They colocalized with markers in the vacuolar membrane, and they also occurred in the plasma membrane in regions of the hyphae more than 1 mm from the tip. The cax gene encodes a Ca2+/H+ exchange protein found in vacuoles. As expected, the CAX protein localized to the vacuolar compartment. We observed, approximately 50 to 100 μm from the tip, a few spherical organelles that had high amounts of tagged CAX protein and tagged subunits of the vacuolar ATPase (VMA-1 and VMA-5). We suggest that this organelle, not described previously in N. crassa, may have a role in sequestering calcium.

scholarly journals AtRBOHC/RHD2 vesicular delivery to the apical plasma membrane domain during root hair development

2021 ◽  
Author(s):  
Lenka Kuběnová ◽  
Michaela Tichá ◽  
Jozef Šamaj ◽  
Miroslav Ovečka
Keyword(s):  
Plasma Membrane ◽  
Quantitative Analysis ◽  
Root Hair ◽  
Tip Growth ◽  
Root Hairs ◽  
Apical Plasma Membrane ◽  
Loss Of Function ◽  
Short Root ◽  
Early Endosomes ◽  
Root Hair Initiation

AbstractArabidopsis root hairs develop as long tubular extensions from the rootward pole of trichoblasts and exert polarized tip growth. The establishment and maintenance of root hair polarity is a complex process involving the local apical production of reactive oxygen species (ROS) generated by NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG PROTEIN C/ROOT HAIR DEFECTIVE 2 (AtRBOHC/RHD2). It has been shown that loss-of-function rhd2 mutants have short root hairs that are unable to elongate by tip growth, and this phenotype was fully complemented by GFP-RHD2 expressed under the RHD2 promoter. However, the spatiotemporal mechanism of AtRBOHC/RHD2 subcellular redistribution and delivery to the plasma membrane (PM) during root hair initiation and tip growth are still unclear. Here, we used advanced microscopy for detailed qualitative and quantitative analysis of vesicular compartments containing GFP-RHD2 and characterization of their movements in developing bulges and growing root hairs. These compartments, identified by an independent marker such as the trans-Golgi network (TGN), deliver GFP-RHD2 to the apical PM domain, the extent of which correlates with the stage of root hair formation. Movements of TGN/early endosomes, but not late endosomes, were affected in the bulging domains of the rhd2-1 mutant. Finally, we reveal that accumulation in the growing tip, docking, and incorporation of TGN compartments containing GFP-RHD2 to the apical PM of root hairs requires structural sterols. These results help clarify the mechanism of polarized AtRBOHC/RHD2 targeting, maintenance, and recycling at the apical PM domain, coordinated with different developmental stages of root hair initiation and growth.One-sentence summaryAdvanced microscopy and quantitative analysis of vesicular TGN compartments revealed that delivering GFP-RHD2 to the apical plasma membrane domains of developing bulges and growing root hairs requires structural sterols.

Integration and regulation of hyphal tip growth

1995 ◽  
Vol 73 (S1) ◽  
pp. 131-139 ◽  
Author(s):  
I. Brent Heath
Keyword(s):  
Cell Wall ◽  
Tip Growth ◽  
Apical Cell ◽  
Turgor Pressure ◽  
Feedback Regulation ◽  
Regulation Mechanism ◽  
Hyphal Tip Growth ◽  
Species Specific ◽  
Stretch Activated Channels ◽  
Hyphal Tip

Hyphal tip growth is an exquisitely controlled process that forms developmentally regulated, species-specific, even-diameter tubes at rates of up to about 50 μm/min. The traditional view is that this process results from the balance between the expansive force of turgor pressure and the controlled extensibility of the apical cell wall. While these elements are involved, the model places regulation into either the global domain (turgor pressure) or the extracellular environment (the cell wall), neither of which seem well suited to the level of control evinced. Recent evidence suggests that F-actin-rich elements of the cytoskeleton are important in tip morphogenesis. Our current models propose that tip expansion is regulated (restrained under normal turgor pressure and protruded under low turgor) by a peripheral network of F-actin that is attached to the plasmalemma and the cell wall by integrin-containing linkages, thus placing control in the cytoplasm where it is accessible to normal intracellular regulatory systems. The F-actin system also functions in cytoplasmic and organelle motility; control of plasmalemma-located, stretch-activated, Ca2+-transporting, ion channel distribution; vectoral vesicle transport; and exocytosis. Regulation of the system may involve Ca2+, the concentration of which is influenced by the tip-high gradient of the stretch-activated channels, thus suggesting a possible feedback regulation mechanism. Key words: tip growth, fungi, stretch-activated channels, F-actin, Ca2+, hyphae.

Integrin and spectrin homologues, and cytoplasm-wall adhesion in tip growth

1995 ◽  
Vol 108 (2) ◽  
pp. 849-856 ◽  
Author(s):  
S.G. Kaminskyj ◽  
I.B. Heath
Keyword(s):  
Growth Rate ◽  
Cell Wall ◽  
Plasma Membrane ◽  
Tip Growth ◽  
Size Range ◽  
Consensus Sequence ◽  
Differential Resistance ◽  
Similar Distribution ◽  
Saprolegnia Ferax ◽  
Beta 1 Integrin

Saprolegnia ferax contains an integrin homologue, identified by crossreactivity with antiserum to the consensus sequence of human/chick/Xenopus cytoplasmic domain beta 1-integrin, which is highly conserved. In non-reduced samples, this integrin was larger than the reported size range for beta 1-integrins, at 178 kDa. In reduced samples, there was a reducing agent-concentration-dependent conversion from 178 kDa to 120 kDa, well within the reported size range for beta 1-integrins in other organisms. The integrin antiserum stained plasma membrane-associated patches, which had a shallow tip-high gradient. This population was reduced and its distribution perturbed in hyphae whose growth rate was reduced by half with tetrapentyl ammonium chloride. The expected integrin function in cytoplasm-cell wall attachment was shown by differential resistance to plasmolysis-induced separation, which positively correlated with integrin abundance. However, when there was separation, remnants of cytoplasm stayed attached to the wall. These were enriched in actin and integrin. Saprolegnia also has a spectrin homologue identified by crossreactivity with an erythrocyte spectin antibody, which has a size (246 kDa) similar to other organisms. This spectrin had a superficially similar distribution to that of integrin, but it did not participate in cytoplasm-wall anchoring. These data suggest that Saprolegnia hyphae have a plasma membrane which is strengthened by spectrin, and cytoplasm which is attached to the cell wall by integrin.

Time series analysis demonstrates the absence of pulsatile hyphal growth

Microbiology ◽  
2003 ◽  
Vol 149 (11) ◽  
pp. 3111-3119 ◽  
Author(s):  
Karina Sampson ◽  
Roger R. Lew ◽  
I. Brent Heath
Keyword(s):  
Growth Rate ◽  
Time Series ◽  
Growth Rates ◽  
Tip Growth ◽  
Hyphal Growth ◽  
Pollen Tubes ◽  
Saprolegnia Ferax ◽  
Hyphal Tip Growth ◽  
Rigorous Statistical Analysis ◽  
Hyphal Tip

Hyphal tip growth has been previously reported as pulsatile, defined as regularly alternating fast and slow rates of extension. The growth of pollen tubes, and hyphae of Neurospora crassa and Saprolegnia ferax were analysed using high spatial and temporal resolution. By using long (100–500 s) records of growth rate, sampled every second, it was possible to apply rigorous statistical analysis of the time series. As previously demonstrated, pollen tubes can show pulsatile growth, detectable with this system. In contrast, hyphal growth rates do not show any evidence of pulsatile growth; instead, growth rates appear to fluctuate randomly. It is concluded that pulsatile growth is not a common feature of hyphal tip growth.

Cytoskeletal regulation of ion channel distribution in the tip-growing organism Saprolegnia ferax

1994 ◽  
Vol 107 (1) ◽  
pp. 127-134 ◽  
Author(s):  
N.N. Levina ◽  
R.R. Lew ◽  
I.B. Heath
Keyword(s):  
Tip Growth ◽  
Actin Network ◽  
Direct Role ◽  
High Gradient ◽  
Saprolegnia Ferax ◽  
Channel Distribution ◽  
Cytoplasmic Actin ◽  
Cytoskeletal Regulation ◽  
Cytochalasin E ◽  
Stretch Activated Channels

Growing hyphal tips of the oomycete Saprolegnia ferax possess a tip-high gradient of stretch-activated ion channels permeable to calcium. These mechanosensitive channels appear to play a direct role in the polarized tip growth process. Treatment of S. ferax hyphae with cytochalasin E leads to the disruption of plasmalemma-associated, peripheral cytoplasmic actin populations and altered morphology of apical protoplasts, and eliminates the tip-high gradient of stretch-activated channels. Cytochalasin E did not alter the normal aggregation of stretch-activated channels. The density of spontaneous K+ channels was decreased in all regions of the hyphae after treatment with cytochalasin E. These results suggest that the peripheral F-actin network in the growing tip of S. ferax hyphae establishes or maintains the tip-high gradient of SA channels, either by the delivery of channel-bearing vesicles to the apex or by the interactions between the channels and the peripheral actin network.

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